The compounds included in the present invention are quinazoline derivatives previously described as inhibitors of tyrosine kinase from the family of EGF receptors (Fry et al., Science 1994, 265, 1093; and Patents No. BR9708640 and EP 566226), now they are unexpectedly found as inhibitors of adenosine kinase.
For a better comprehension of the present invention, some known compounds and their properties are defined and described, together with specific terms related to this invention.
Adenosine. Adenosine is a purinic nucleoside that regulates multiple cell functions, and its effects are mediated by at least four kinds of P1 purinergic receptors located in cell membranes of almost all kinds of cells, namely A1, A2a, A2b and A3 (Fredholm et al., Pharmacol. Rev. 2001, 53:527). Despite acting in practically in all cells and organs of the organism, its main effects are observed in the heart, brain, kidneys and immune system cells. Because its action is restricted to the site where it is released, adenosine is considered an autacoid (form the Greek autos—self and akos—relief, cure). In general, its complex effects result in reduction of metabolic activity and protection against physiological and pathological stimuli associated to sustained increases of cell activity. Its protective effects are well known for ischemia/reperfusion deleterious actions, pro-inflammatory substances, as well as its effects as analgesic, modulator of neuronal activity related to sleep, sympatholitic, inhibitor of thrombosis aggregation, inhibitor of neutrophilic adhesion, inhibitor of free radical production and vasodilator.
Pharmacological inhibitors of the adenosine catabolism. Considering its wide range of effects, there is no surprise in the rising interests in the therapeutic usage of adenosine, its mimetics and in substances that interfere in its metabolism and bioavailability. The usage of adenosine itself as a therapeutic agent is restricted due to its short half-life (estimated in less than one second in circulation) and its undesirable hemodynamic effects. These arguments are also valid for agonists and antagonists of receptor that like adenosine may have undesirable systemic effects. However, approaches that interfere in local metabolism and bioavailability of adenosine are promising. In this case, it should be noticed that adenosine is formed mainly as a result of the action of intra and extra-cellular 5′-nucleotidases that dephosphorylate 5′-AMP, and of the SAH-hydrolase on the S-adenosyl-homocysteinee (SAH) (Headrick et al., Am. J. Physiol. Heart Circ. Physiol. 2003, 285:H1797). Otherwise, extra-cellular adenosine is quickly absorbed by cells through a specific membrane carrier. In the intracellular environment, adenosine is deaminated turning into inosine, by adenosine deaminase or re-phosphorylated into 5′-AMP by adenosine kinase. The high catabolism, besides the high membrane transport speed, determines that adenosine has a short half-life and highly localized physiological functions. The importance of these mechanisms for local adenosine bioavailability is confirmed by the increase in tissue concentration caused by inhibitors of membrane carriers of adenosine, as well as by the action activity inhibitors of adenosine kinase or deaminase (Headrick et al., Am. J. Physiol. Heart Circ. Physiol. 2003, 285:H1797; and Kowaluk and Jarvis, Expert Opin. Investig. Drugs 2000, 9:551). In this case of enzyme inhibitors, available evidences suggest a potential utility for the therapy of clinical situations, where deleterious effects of ischemia/reperfusion, inflammation and pain are involved.
Adenosine Kinase. Adenosine kinase, also known as adenosine 5′-phosphotransferase, is the most abundant nucleoside kinase in mammals, and catalyzes the phosphorylation of the 5′-hydroxyl of the ribofuranosyl of nucleoside analogs, using ATP or GTP as phosphate donor. The structures of adenosine kinases from different species, including human, have been determined, obtained from the placenta. The enzyme is a monomer, whose structure consists of a large α/β dominion with nine β-bands and eight α-helixes and a smaller α/β dominion with five β-bands and two α-helixes (Mathews et al., Biochemistry 1998, 37:15607). The active site is located along the edge of the β-band in the larger α/β dominion, and this is where adenosine attaches itself, while the smaller α/β dominion blocks the upper face of the active site, and another nearby site receives the ATP. A magnesium binding site is located between binding sites of adenosine and ATP, and this is an essential ion for the catalysis of adenosine kinase. The model proposed for the activity of this kinase suggests that the amino acid aspartate, located in the position 300, is an important catalytic remainder involved in the deprotonation of the 5′-hydroxyl group during phosphate transfer.
The pharmacological inhibition of adenosine kinase has been described with adenosine analogs (e.g., aminoadenosine and iodotubercidine), as well as with pyridopyrimidine derivatives (Kowart et al., Bioorg. Med. Chem. Lett. 2001, 11:83; Lee et al., J. Med. Chem. 2001, 44:2133; Zheng et al., Bioorg. Med. Chem. Lett. 2001, 11:2071; Gomitsian et al., J. Med. Chem. 2002, 45:3639; Gfesser et al., Eur. J. Med. Chem. 2003, 38:245; Zheng et al., Bioorg. Med. Chem. Lett. 2003, 13:3041; and Perner et al., J. Med. Chem. 2003, 46:5249). Studies of therapeutic application of these compounds have shown beneficial effects of inhibition of the adenosine kinase in situations of myocardium ischemia, pain and inflammatory processes (Jarvis et al., Pain 2002, 96:107; Suzuki et al., Br. J. Pharmacol. 2001, 132:1615; Boyle et al., J. Pharmacol. Exp. Ther. 2001, 296:495; Kowaluk et al., J. Pharmacol. Exp. Ther. 2000, 295:1165; Jarvis et al., J. Pharmacol. Exp. Ther. 2000, 295:1156; and Smolenski et al., Circulation 2001, 104(suppl I):I-246).
Ischemia/Reperfusion. Ischemia, defined as improper blood supply to tissues and organs, is one of the main causes of death and disability in populations all over the world, and its main determinant is the atherosclerotic disease of arteries. Its effects in the heart, brain or kidneys are caused mainly by the lack of oxygen, which leads to, depending on intensity and duration, to death or cell degeneration, resulting in different clinical situations like myocardium infarct, chest angina, heart insufficiency, brain vascular accident and kidney insufficiency. Otherwise, an additional deleterious effect is caused after restoration of blood flow in the ischemic area, a condition that many times occurs spontaneously or by therapeutic interference (e.g., coronary thrombosis). Mechanisms that cause deleterious effects of reperfusion are poorly known. Nevertheless, important pathogenic agents considered are the massive oxy radicals generation and the overload of intracellular calcium due to reperfusion. Therefore, tissue lesions caused by ischemia are frequently a consequence of a combination of deleterious effects of the ischemia per se and of reperfusion. We should include in the context lesions to organs (e.g., heart, kidney and liver) used in transplantations.
Surely, the obvious solution to the morbid-mortality caused by conditions of ischemia in several organs is the prevention of atherosclerotic disease. However, the impact of available strategies for primary prevention is still very limited. Therefore, effective prevention ways are needed and, particularly, therapeutic ways to limit the extension of tissue lesions caused by ischemia, and the preservation of the viability of ischemic tissues is one of the most imperious present therapeutic objectives.
In this context, it is important to mention that cells from multicellular organisms have a self-protection mechanism for the lesion by ischemia/reperfusion, activated by repeated events of sub-lethal ischemia, known as ischemic pre-conditioning (Yellon and Downey, Physiol. Rev. 2003, 83:1113). This mechanism has two ways of protection: one known as “classical” that last about two hours after the conditioning ischemia, followed after about 24 hours by a second protection window that lasts three days, known as “late protection.” The current model for explaining preconditioning states that conditioning ischemia causes the release of various autacoids that trigger the protection process through the activation of membrane receptors (Yellon and Downey, Physiol. Rev. 2003, 83:1113). This activation triggers the combination of complex cell signaling ways that during lethal ischemia converge to one or more effectors to mediate protection. The effectors of this response are still poorly known. Nevertheless, in the therapeutic point-of-view, it is important that pharmacological agents that activate signaling ways at different levels may mimic the conditioning stimulus, leading to the expectation that pharmacological agents may be produced in order to explore therapeutically the powerful tissue protection activated by endogenous mechanisms responsible to ischemic pre-conditioning.
Thus, it is known that adenosine is the main triggering agent in the activation of cell ways involved in the classical or late pre-conditioning (Headrick et al., Am. J. Physiol. Heart Circ. Physiol. 2003, 285:H1797). Consistent results from clinical studies point out benefits of the use of adenosine for the preservation of the ischemic myocardium, but clinical evidences are still scarce for its therapeutic effect in brain and kidney ischemic lesions, yet it does not mean that it is not effective. It has been demonstrated, for instance, that its action restores ATP stocks in endothelium cells and myocytes, inhibits the formation of free radicals, inhibits the accumulation and the activity of neutrophils, and improves microcirculation (Mahaffey et al., J. Am. Col. Cardiol. 1999, 34:1711). Additionally, because adenosine is the main endogenous agent that activates ischemic pre-conditioning, its effect is particularly important in acute coronary syndromes, for they are usually caused by dynamic coronary occlusion with intermittent blood flow periods, having a potential deleterious effect due to the ischemia/reperfusion mechanism. In models of acute coronary syndrome in experimental animals, adenosine reduces consistently the size of the infarct, improves ventricular function and improves coronary flow (Yellon and Downey, Physiol. Rev. 2003, 83:1113; and Headrick et al., Am. J. Physiol. Heart Circ. Physiol. 2003, 285:H1797). Clinical studies demonstrated that adenosine administration reduces the extension of myocardium infarcts, improves the conditions of myocardium flow, reduces the incidence of heart insufficiency and of myocardium infarct with Q wave in patients submitted to primary angioplasty, also reduces variation of the segment S-T, lactate production and ischemic symptoms in patients submitted to elective angioplasty (Mahaffey et al., J. Am. Col. Cardiol. 1999, 34:1711). Recently, results from the study AMISTAD (Acute Myocardial Infarction Study of Adenosine), planned to test the hypothesis that adenosine reduces the size of myocardium infarct in patients submitted to thrombolysis, demonstrated reduction in sizes of previous infarcts in patients treated with adenosine (Mahaffey et al., J. Am. Col. Cardiol. 1999, 34:1711). However, no differences between the clinical evolution of treated and non-treated patients was observed. The absence of measurable clinical benefits with adenosine in this study reflects problems with biases in choice of patient groups, but also pharmacokinetic and pharmacodynamic problems of adenosine, as well as its short half-life and undesirable hemodynamic effects.
Therefore, it is possible that pharmacological agents that modify local adenosine bioavailability show to be effective for the protection of the myocardium and other tissues submitted to ischemia/reperfusion.
Inflammation. Chronic inflammatory diseases represent a wide range of diseases that attack organs and tissues in different ways and extensions. In this group, one may include, among others, asthma, rheumatoid arthritis, inflammatory diseases of the intestine, psoriasis and atherosclerosis (Barnes and Karim, N. Engl. J. Med. 1997, 336:1066; and Ross, N. Engl. J. Med. 1999, 340:115). In spite of representing different physiopathological situations, all inflammatory diseases present the activation and collapse of the immune system responsible for the amplification and support of the inflammatory process. Causes of these diseases remain unknown, but there is little doubt that the pathological process results from the interaction between genetic and environmental factors. Genes, like those in asthma atopy, HLA antigens in rheumatoid arthritis and intestine inflammatory diseases, may determine the susceptibility of patients to the disease, but frequently unknown environmental factors may determine clinical presentation and course. Once established, the chronic inflammatory process develops itself alone. Anti-inflammatory agents and immunosuppressors may suppress the vicious circle, but there is still no healing treatment for any chronic inflammatory diseases.
Deleterious effects of chronic inflammatory processes occur through several mechanisms, but main determinants are local production of pro-inflammatory cytokines and transformation of tissue inflammatory cells in autonomous lineages. These transformations and cytokine production are processes regulated by complex signaling ways that involve many transduction elements and transcription factors. Nevertheless, one transcription factor, NF-κB, seems to be a key element for the activation and transformation of tissue inflammatory cells (Barnes and Karim, N. Engl. J. Med. 1997, 336:1066; and Lawrence et al., Nat. Med. 2001, 7:1291). This factor is related to the expression of genes responsible for adhesion and recruitment of circulation inflammatory cells (e.g., neutrophils, eosinophils and T lymphocytes) in inflammatory sites, as well as for cytokine and enzyme production in chronic inflammatory diseases. One of these genes is the inducible NOS, whose expression in increased in the epithelium of aerial ways cells and macrophages of asthma patients, in colon epithelium of ulcerative colitis patients and in synovial cells of inflamed joints. The cyclooxygenase-2, another inducible enzyme regulated by NF-κB, is responsible by the increase in prostaglandin and tromboxane production in inflammatory diseases. On the other hand, the production of interleukin-1β, TNF-α, interleukin-6, stimulant factor of granulocyte/macrophage colonies, and many chemotactic cytokines is increased in patients of asthma, rheumatoid arthritis, psoriasis and intestine inflammatory disease. All these cytokines have an important role in these inflammatory processes. Interleukin-1β and TNF-α may influence the severity of these diseases, possibly by permanently activating NF-κB. The treatment of rheumatoid arthritis patients with drugs that block the action of TNF-α may control the disease.
Adenosine is an endogenous immunomodulator with anti-inflammatory and immunosuppressor properties, which acts through multiple mechanisms still not completely established. Some evidences point out that adenosine inhibits the activation of NF-κB that is induced by TNF, what may contribute for its role in the suppression of inflammations and immunomodulation (Kowaluk et al., J. Pharmacol. Exp. Ther. 2000, 295:1165; and Jarvis et al., J. Pharmacol. Exp. Ther. 2000, 295:1156). Therefore, the use of adenosine kinase inhibitors may present therapeutic benefits to a wide range of clinical situations directly or indirectly dependent of inflammatory and immunological processes. Among conditions that could benefit from the use of adenosine kinase are chronic degenerative inflammatory diseases (e.g., rheumatoid arthritis, systemic erythematous lupus etc.), asthma, atherosclerosis, ulcerative colitis, and Chron disease.
Pain. Chronic or acute pain are among most frequent clinical conditions. Mechanisms involved in its beginning and sustenance are multiple and comprise from neuronal degeneration to inflammation. Pain initiator stimuli are transmitted to the central nervous system by the activation of non-myelinized (C fibers) and myelinized (Aδ fibers) afferents. Cell bodies of these fibers are located in the dorsal root, trigeminal ganglion, and nodous ganglion. These fibers establish multiple connections with the spinal medulla or cerebral trunk, and with specific areas of the prosencephalon, where the stimulus is integrated. Following the tissue lesion or the inflammation, a large number of endogenous substances are released, and these substances may activate or sensibilize nociceptor afferents. These substances comprise H+, ATP, bradikinine, 5-HT, histamine, prostaglandins, P substance and adenosine (Bevan, 1999, In: P. D. Wall and R. Melzack (Eds.), Textbook of Pain, fourth ed. Churchill Livingstone, Edinburgh, pp. 85-103). Some of these mediators act through binders associated to cationic channels (e.g., H+, ATP, 5-HT3), while others act through G-protein-coupled receptors (GPCRs) (e.g., prostaglandins, bradikinines, 5-HT). Changes in the excitability of nociceptor afferents may result from the activation of multiple intracellular signaling ways mediated by kinase proteins with subsequent phosphorylation of specific sodium channels of sensorial neurons. There are three basic therapeutic approaches for controlling pain: (1) suppression of the source, (2) change in central perception, and (3) transmission blocking for the central nervous system.
Adenosine and its analogs have analgesic effect. Their actions are complex and multiple, including action in central and peripheral mechanisms. Thus, spinal administration of adenosine or its analogs (e.g., 5′-N-ethyl-carboxamidoadenosine (NECA)) produces analgesia through an effect mediated by A1 receptors, whose activation produces liberation inhibition of nociceptive afferents CGRP (Mauborgne et al., Eur. J. Pharmacol. 2002, 441:47). Likewise, the same effect has been demonstrated for adenosine metabolism inhibitors (Sawynok, Curr. Opin. Cent. Periph. Nerv. Syst. Invest. Drugs 1999, 1:27; and Kowaluk et al., Exp. Opin. Invest. Drugs 2000, 9:551). The inhibition of adenosine kinase with 5′-amino-5′-deoxyadenosine or iodotubercidine increase the bioavailability of adenosine in the spinal medulla (Golembiowska et al., Brain Res. 1995, 699:315).
Adenosine acts also directly on peripheral nerves by interfering in the process of nociceptor activation, through complex mechanisms. Its actions may result in inhibition or increase of pain through the action on nociceptor afferents via A1 and A2A receptors, and it results from the reduction or increase of cAMP, respectively (Khasar et al., Neuroscience 1995, 67:189). However, its central actions are more powerful and result in analgesic effect.
Anilinoquinazolines: Derivatives of 4-anilinoquinazolines are widely described in the literature as powerful and selective inhibitors of the activity of tyrosine kinases from the family of EGF receptors (Fry et al., Science 1994, 265, 1093; Fry et al., Pharmacol Ther. 1999, 82, 207; and Levitzki et al., Pharmacol. Ther. 1999, 82, 231). Furthermore, knowledge of the inhibition process of these enzymes seems to be the way for the therapy of many diseases, like cancer, psoriasis, diabetes, cardiovascular diseases etc (Fry et al., Science 1994, 265, 1093). Based on this evidence, many detailed studies arose on the biological function of many derivatives from this structure class (Rewcastle et al., J. Med. Chem. 1995, 38, 3482; and Bridges et al., J. Med. Chem. 1996, 39, 267).
Many studies on the structure-activity relationship (SAR) involving many series of quinazoline derivatives lead to advances in power, specificity and pharmacokinetic properties of these inhibitors (Fry et al., Pharmacol. Ther. 1999, 82, 207; and Rewcastle et al., Curr. Org. Chem. 2000, 4, 679). Three quinazoline compounds are under clinical investigation in cancer patients: ZD1839 (Iressa) (Rewcastle et al., Curr. Org. Chem. 2000, 4, 679), CP358774 (Rewcastle et al., Curr. Org. Chem. 2000,4, 679; and Moyer et al., Cancer Res. 1997, 57, 4838) and CI1033 (Tsou et al., J. Med. Chem. 2001, 44, 2719). Pre-clinical data (IC50 in the order of pmol.L−1) support the possibility of using these compounds in conventional chemotherapy with potential anti-tumoral agents (Ciardiello et al., Drugs 2000, 60 (supl. 1), 25).
Inhibition power, in all series of evaluated and synthesized compounds, seems to be associated to electron donor substitute groups in positions 6 and/or 7 of quinazoline (OMe, OEt e NH2), and to halogens (mainly Br and Cl), like substitutes in the meta position of the aniline ring. The meta-substituted aniline group showed to be the best substitute for position 4 of the quinazoline system (Bridges et al., J. Med. Chem. 1996, 39, 267).
Studies with quinazoline derivatives have not been limited only to the investigation of the activity of tyrosine kinase from the family of EGF receptor (Rewcastle et al., Curr. Org. Chem. 2000, 4, 679). Prazosin is a quinazoline with antagonistic properties of α-adrenergic receptors. This compound has a vasodilator effect, and is used in the anti-hypertensive therapy, as well as some of its structural derivatives like ciclazosin, which has a stringer affinity to α1-adrenergic receptors, and may be applied in the treatment of benign prostate hyperplasia (Melchiorre et. al., Bioorganic and Medicinal Chemistry Letters 1998, 8, 1353-1358). Another good example is PD153035, which entered the stage of clinical triage by Sugen (like SU5271) for use in the treatment of skin diseases, like psoriasis and skin cancer (McMahon et al., WO9810767; Chem. Abstr. 1998, 128, 261949). Other examples of biologically active quinazolines are those presented as powerful and specific inhibitors of type 5 phosphodiesterase (PDE5) (Ukita et al., J. Med. Chem. 2001, 44, 2204). This enzyme is highly specific in the hydrolysis of the cyclic nucleotide cGMP (guanosine 3′,5′-cyclic monophosphate), which controls vascular functions (Corbin et al., J. Biol. Chem. 1999, 274, 13729). Thus, an inhibitor that increases the cGMP level inside cells is considered a potential pharmaceutical for the treatment of cardiovascular diseases, such as hypertension, angina, and heart insufficiency (Ukita et al., J. Med. Chem. 2001, 44, 2204).